Abstract
Regulation of intracellular Ca2+ concentration ([Ca 2+]i) is a key factor in airway smooth muscle (ASM) tone. In vascular smooth muscle, specialized membrane microdomains (caveolae) expressing the scaffolding protein caveolin-1 are thought to facilitate cellular signal transduction. In human ASM cells, we tested the hypothesis that caveolae mediate Ca2+ responses to agonist stimulation. Fluorescence immunocytochemistry with confocal microscopy, as well as Western blot analysis, was used to determine that agonist receptors (M3 muscarinic, bradykinin, and histamine) and store-operated Ca2+ entry (SOCE)-regulatory mechanisms colocalize with caveolin-1. Although caveolin-2 coexpressed with caveolin-1, caveolin-3 was absent. In fura 2-loaded ASM cells, [Ca2+]i responses to 1 μM ACh, 10 μM histamine, and 10 nM bradykinin, as well as SOCE, were attenuated (each to a different extent) after disruption of caveolae by the cholesterol-chelating drug methyl-β-cyclodextrin. Transfection of ASM cells with 50 nM caveolin-1 small interfering RNA significantly weakened caveolin-1 expression and blunted [Ca2+]i responses to bradykinin and histamine, as well as SOCE, but the response to ACh was less intense. These results indicate that caveolae are present in ASM and that caveolin-1 contributes to regulation of [Ca2+]i responses to agonist.
| Original language | English (US) |
|---|---|
| Journal | American Journal of Physiology - Lung Cellular and Molecular Physiology |
| Volume | 293 |
| Issue number | 5 |
| DOIs | |
| State | Published - Nov 2007 |
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Keywords
- Intracellular signaling pathway
- Methyl-β-cyclodextrin
- RNA
- Small interfering
- Store-operated calcium entry
ASJC Scopus subject areas
- Cell Biology
- Physiology
- Pulmonary and Respiratory Medicine
Cite this
Caveolins and intracellular calcium regulation in human airway smooth muscle. / Prakash, Y.s.; Thompson, Michael A.; Vaa, Brianna; Matabdin, Ihaab; Peterson, Timothy E.; He, Tongrong; Pabelick, Christina M.
In: American Journal of Physiology - Lung Cellular and Molecular Physiology, Vol. 293, No. 5, 11.2007.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Caveolins and intracellular calcium regulation in human airway smooth muscle
AU - Prakash, Y.s.
AU - Thompson, Michael A.
AU - Vaa, Brianna
AU - Matabdin, Ihaab
AU - Peterson, Timothy E.
AU - He, Tongrong
AU - Pabelick, Christina M
PY - 2007/11
Y1 - 2007/11
N2 - Regulation of intracellular Ca2+ concentration ([Ca 2+]i) is a key factor in airway smooth muscle (ASM) tone. In vascular smooth muscle, specialized membrane microdomains (caveolae) expressing the scaffolding protein caveolin-1 are thought to facilitate cellular signal transduction. In human ASM cells, we tested the hypothesis that caveolae mediate Ca2+ responses to agonist stimulation. Fluorescence immunocytochemistry with confocal microscopy, as well as Western blot analysis, was used to determine that agonist receptors (M3 muscarinic, bradykinin, and histamine) and store-operated Ca2+ entry (SOCE)-regulatory mechanisms colocalize with caveolin-1. Although caveolin-2 coexpressed with caveolin-1, caveolin-3 was absent. In fura 2-loaded ASM cells, [Ca2+]i responses to 1 μM ACh, 10 μM histamine, and 10 nM bradykinin, as well as SOCE, were attenuated (each to a different extent) after disruption of caveolae by the cholesterol-chelating drug methyl-β-cyclodextrin. Transfection of ASM cells with 50 nM caveolin-1 small interfering RNA significantly weakened caveolin-1 expression and blunted [Ca2+]i responses to bradykinin and histamine, as well as SOCE, but the response to ACh was less intense. These results indicate that caveolae are present in ASM and that caveolin-1 contributes to regulation of [Ca2+]i responses to agonist.
AB - Regulation of intracellular Ca2+ concentration ([Ca 2+]i) is a key factor in airway smooth muscle (ASM) tone. In vascular smooth muscle, specialized membrane microdomains (caveolae) expressing the scaffolding protein caveolin-1 are thought to facilitate cellular signal transduction. In human ASM cells, we tested the hypothesis that caveolae mediate Ca2+ responses to agonist stimulation. Fluorescence immunocytochemistry with confocal microscopy, as well as Western blot analysis, was used to determine that agonist receptors (M3 muscarinic, bradykinin, and histamine) and store-operated Ca2+ entry (SOCE)-regulatory mechanisms colocalize with caveolin-1. Although caveolin-2 coexpressed with caveolin-1, caveolin-3 was absent. In fura 2-loaded ASM cells, [Ca2+]i responses to 1 μM ACh, 10 μM histamine, and 10 nM bradykinin, as well as SOCE, were attenuated (each to a different extent) after disruption of caveolae by the cholesterol-chelating drug methyl-β-cyclodextrin. Transfection of ASM cells with 50 nM caveolin-1 small interfering RNA significantly weakened caveolin-1 expression and blunted [Ca2+]i responses to bradykinin and histamine, as well as SOCE, but the response to ACh was less intense. These results indicate that caveolae are present in ASM and that caveolin-1 contributes to regulation of [Ca2+]i responses to agonist.
KW - Intracellular signaling pathway
KW - Methyl-β-cyclodextrin
KW - RNA
KW - Small interfering
KW - Store-operated calcium entry
UR - http://www.scopus.com/inward/record.url?scp=36348971329&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=36348971329&partnerID=8YFLogxK
U2 - 10.1152/ajplung.00136.2007
DO - 10.1152/ajplung.00136.2007
M3 - Article
C2 - 17704188
AN - SCOPUS:36348971329
VL - 293
JO - American Journal of Physiology - Renal Fluid and Electrolyte Physiology
JF - American Journal of Physiology - Renal Fluid and Electrolyte Physiology
SN - 1931-857X
IS - 5
ER -